Polymerase‐Mediated Site‐Specific Incorporation of a Synthetic Fluorescent Isomorphic G Surrogate into RNA

Li, Yao; Fin, Andréa; McCoy, Lisa S.; Tor, Yitzhak

doi:10.1002/ange.201609327

Cited by 14 publications

(6 citation statements)

References 65 publications

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“…The equilibrium between these tautomers informs on the local environment of th G. Due to its unique properties, th G was successfully used to monitor the local conformational changes associated to the binding of the HIV-1 nucleocapsid protein NCp7 to the (−)Primer Binding Site (PBS) and to decipher the mechanism of the NCp7-promoted (+)/(−)PBS annealing reaction 7 . The fluorescent nucleoside analogue in its deoxyribo or in its ribo form has also been successfully used to monitor B to Z transitions of DNA 2 , single nucleotide polymorphism 7 , distance measurements in DNA 8,9 , base flipping reactions 10 , ribozyme-mediated cleavage 11 , and cellular activity of siRNAs 12 .…”

Section: Introductionmentioning

confidence: 99%

What Makes Thienoguanosine an Outstanding Fluorescent DNA Probe?

et al. 2020

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Thienoguanosine ( th G) is an isomorphic guanosine (G) surrogate that almost perfectly mimics G in nucleic acids. To exploit its full potential and lay the foundation for future applications, twenty DNA duplexes, where the bases facing and neighboring th G were systematically varied, were thoroughly studied using fluorescence spectroscopy, molecular dynamics simulations and mixed quantum mechanical/molecular mechanics calculations, yielding a comprehensive understanding of its photophysics in DNA. In matched duplexes, th G's hypochromism was larger for flanking G/C residues but its fluorescence quantum yield (QY) and lifetime values were almost independent of the flanking bases. This was attributed to high duplex stability, which maintains a steady orientation and distance between nucleobases, so that a similar charge transfer (CT) mechanism governs the photophysics of th G independently of its flanking nucleobases. th G can therefore replace any G residue in matched duplexes, while always maintaining similar photophysical features. In contrast, the local destabilization induced by a mismatch or an abasic site restores a strong dependence of th G's QY and lifetime values on its environmental context, depending on the CT route efficiency and solvent exposure of th G. Due to this exquisite sensitivity, th G appears ideal for monitoring local structural changes and single nucleotide polymorphism.Moreover, th G's dominant fluorescence lifetime in DNA is unusually long (9-29 ns), facilitating its selective measurement in complex media using a lifetime-based or a time-gated detection *

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Section: Introductionmentioning

confidence: 99%

What Makes Thienoguanosine an Outstanding Fluorescent DNA Probe?

et al. 2020

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“…Herein, as an alternative approach, we report the enzymatic synthesis of DNA strands containing a hydroxypyridone ligand-type artificial nucleotide (H), which forms a stable Cu II -mediated unnatural base pair (H−Cu II −H) 21−26 (Figure 1). Enzymatic incorporation of unnatural nucleotides, 27,28 including ligand-type nucleotides, 25,26,29−34 has recently attracted increasing attention as a powerful method to prepare functional nucleic acids. In contrast to the conventional solidphase DNA synthesis, enzymatic reactions are expected to proceed under mild conditions without any additional protecting groups on metal coordinating functionalities.…”

Section: ■ Introductionmentioning

confidence: 99%

Enzymatic Synthesis of Cu(II)-Responsive Deoxyribozymes through Polymerase Incorporation of Artificial Ligand-Type Nucleotides

2019

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Metal-mediated artificial base pairs, consisting of ligand-type nucleotides and a bridging metal ion, have shown promise as functional units to develop stimuli-responsive DNA materials. Although a variety of metal-mediated base pairs have been constructed with artificial ligand-type nucleotides and various metal ions, the application of such metal-mediated base pairs has been relatively poorly explored mainly due to the cumbersome chemical synthesis of artificial DNA strands. Herein we report a facile enzymatic method to synthesize DNA strands containing a ligand-type hydroxypyridone (H) nucleotide, which forms a Cu II -mediated base pair (H−Cu II −H). A two-step primer extension reaction using two commercially available polymerases enabled the incorporation of a H nucleotide at an internal position of oligonucleotides. The polymerase synthesis was subsequently applied to the development of metal-responsive deoxyribozymes (DNAzymes), whose catalytic activity was regulated by the formation of a single H−Cu II − H base pair in its stem region. The DNAzyme activity was reversibly switched by the alternate addition and the removal of Cu II ions. Furthermore, metal-dependent orthogonal activation of a Cu II -responsive H-DNAzyme and a Hg II -responsive T-DNAzyme was experimentally demonstrated by utilizing both H−Cu II −H as well as widely explored T−Hg II −T base pairs. These results suggest that the incorporation of H−Cu II −H base pairs would facilitate the rational design of metal-responsive functional DNAs. Accordingly, the facile enzymatic synthesis of artificial ligand-bearing DNAs developed in this study would significantly expand the toolbox of DNA-based supramolecular chemistry and DNA nanotechnology.

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“…To overcome these shortcomings, we sought for an alternative G surrogate that could more effectively mimic natural GTP in RZA synthesis, while being compatible with the other three modified rNTPs (rZTPs) mentioned above. We examined thienoguanosine triphosphate ( th GTP, Figure 1), which is known to bear a high resemblance to natural GTP and act as a substrate for T7 RNAP during both initiation and elongation phases (16,17), while possessing unique photophysical properties including visible emission, high quantum yield, and responsiveness to environmental perturbations (18)(19)(20)(21). While short th G-modified RNA oligonucleotides have been previously produced by solidphase synthesis for various biophysical applications (22)(23)(24), only a few th G modifications were incorporated at the initial site or specific positions.…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids

Yang

Eremeeva

Abramov

et al. 2023

Nucleic Acids Research

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An enzymatic method has been successfully established enabling the generation of partially base-modified RNA (previously named RZA) constructs, in which all G residues were replaced by isomorphic fluorescent thienoguanosine (thG) analogs, as well as fully modified RZA featuring thG, 5-bromocytosine, 7-deazaadenine and 5-chlorouracil. The transcriptional efficiency of emissive fully modified RZA was found to benefit from the use of various T7 RNA polymerase variants. Moreover, dthG could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing thG as well as thG together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N1-Methylpseudouridine was also demonstrated to be a faithful non-canonical substitute of uridine to direct Cas9 nuclease cleavage when incorporated in sgRNA. The Cas9 inactivation by 7-deazapurines indicated the importance of the 7-nitrogen atom of purines in both sgRNA and PAM site for achieving efficient Cas9 cleavage. Additional aspects of this study are discussed in relation to the significance of sgRNA–protein and PAM–-protein interactions that were not highlighted by the Cas9–sgRNA–DNA complex crystal structure. These findings could expand the impact and therapeutic value of CRISPR-Cas9 and other RNA-based technologies.

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Polymerase‐Mediated Site‐Specific Incorporation of a Synthetic Fluorescent Isomorphic G Surrogate into RNA

Cited by 14 publications

References 65 publications

What Makes Thienoguanosine an Outstanding Fluorescent DNA Probe?

What Makes Thienoguanosine an Outstanding Fluorescent DNA Probe?

Enzymatic Synthesis of Cu(II)-Responsive Deoxyribozymes through Polymerase Incorporation of Artificial Ligand-Type Nucleotides

CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids

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